菲科尔
鹌鹑
生殖系
生物
同源盒蛋白纳米
Percoll公司
干细胞
移植
分子生物学
在ovo
细胞生物学
嵌合体(遗传学)
男科
遗传学
胚胎
基因
体外
胚胎干细胞
诱导多能干细胞
内科学
内分泌学
医学
外周血单个核细胞
作者
Jae Yong Han,Ho Yeon Cho,Young Min Kim,Kyung Je Park,Kyung Min Jung,Jin Se Park
标识
DOI:10.1016/j.theriogenology.2020.05.039
摘要
Due to the absence of long-term in vitro germline competent stem cell maintenance systems and efficient methods for germline transmission, efforts to develop an effective transgenic system in quail has remained limited. To overcome this limitation, here we produced germline chimeric quails through transplantation of spermatogonial stem cells (SSCs) enriched by density gradient methods utilizing Ficoll-Paque PLUS (Ficoll), Percoll and sucrose solution as a practical strategy for germline transmission in quail. For all gradient methods, testicular cells were separated as two fractions, and the expression levels of SSC-specific genes (GFRA1, ITGA6, ITGB1) and pluripotency genes (NANOG, POUV) were examined. As a result, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and RNA probe hybridization analysis revealed that the upper fraction that was separated by Ficoll showed the highest expression of SSC-specific and pluripotency genes among all fractions. Cells in the upper Ficoll gradient fraction also displayed reduced heterochromatin distribution, as observed in differentiated spermatogonia using transmission electron microscopy (TEM). These results indicate that SSCs were enriched in the upper fraction by Ficoll density gradient centrifugation. Subsequent transplantation experiments revealed that the efficiency of germline transmission to donor-derived gametes in the germline chimeras with transplanted SSCs and whole testicular cells was 0–13.2% and 0–4.4%, respectively. Collectively, these results demonstrate that quail SSCs were easily enriched with a density gradient method and that this method is a feasible and practical way to preserve the germplasm of quail. Furthermore, we can expect to apply this method in research examining the production of transgenic quail and preservation of avian species.
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